PROJECT SUMMARY Intracellular pathogens account for a significant amount of morbidity and mortality world-wide. Here, the model facultative intracellular pathogen Listeria monocytogenes (Lm) will be used to investigate how bacterial pathogens recognize and adapt to the host environment. Previous studies suggested a model in which specific alterations in the redox environment are one of the biological cues detected by intracellular pathogens during infection as a mechanism to sense their localization and regulate genes accordingly. Specifically, we identified the redox-responsive transcriptional regulator SpxA1 as essential for aerobic growth in vitro and critical for Lm virulence. Preliminary data revealed that SpxA1 regulates hundreds of genes in vitro and in vivo. These results have set the stage for investigations into the specific genes that are required in each distinct growth environment. Experiments proposed in Aim 1a will identify the genes that are required for Lm aerobic growth in vitro, while Aim 1b and 1c will define the SpxA1-dependent genes required for pathogenesis. Experiments described in Aim 2 will develop reporter strains with which to monitor SpxA1 activity and will apply these to define the host signals that activate SpxA1 in vivo. Professional pathogens, such as Lm, have evolved to resist or evade host-derived antimicrobial factors that target invading pathogens. We will use SpxA1-mediated transcriptional adaptation as a sensitive readout with which to investigate these host defenses. Results from these studies will identify the host cell stressors that are encountered during infection and the corresponding Lm transcriptional response that is required for pathogenesis. A thorough understanding of the signaling cascades that are activated during infection and the host cues that stimulate these pathways may reveal fundamental features of the host cytosol that intracellular bacterial pathogens have evolved to detect.